Sustained increases in vascular permeability, a defining feature ventilator-associated lung injury and multi-organ failure, prolongs the duration of physiologic derangement and the requirement for mechanical ventilation. Increased ventilator days enhance the risk of malnutrition and nosocomial/ventilator-associated pneumonia. Clearly there is a desperate need for new strategies to reduce vascular leak in patients receiving ventilation. Project 4 will utilize well-established models of murine and canine VALI to identify novel molecular targets and validate recently described molecular targets involved in VALI- induced edema formation. Importantly, we will test novel anti-edema strategies which target the endothelial cell cytoskeletal, a direct result of in vitro physiologic, biophysical, biochemical and molecular studies conducted by the Project Leader. SA #1 will test the effect of molecular strategies to reduce the activity of the Ca+2/calmodulin-dependent myosin light chain kinase in lung endothelium (EC MLCK), a critical cytoskeletal regulatory enzyme first cloned by the Garcia laboratory. EC MLCK is intimately involved in multiple aspects of the inflammatory response and directly participates in EC barrier regulation. SA #2 will examine the efficacy of sphingosine 1-phosphate (Sph 1-P), angiogenic factor critical to platelet-mediated vascular integrity, as novel therapy in animal models of ALl. We demonstrated that Sph 1-P produces rapid, sustained, and dose-dependent increases in the barrier integrity of in vitro and in vivo. SA #3 will utilize the lipid-lowering HMG Co A reductase inhibitor, simvastatin, to reduce VALI-induced edema formation. Recent reports including our own data, which indicate that the statins directly affect vascular remodeling, likely via the modulation of intracellular signaling mediated by Rho GTPases and Rho kinase, a pathway utilized by edemagenic agents (such as thrombin and VALI) to increase vascular leak. Finally, the identification of novel therapeutic targets for future barrier- protective strategies is essential for progress to be made in this devastating disorder. SA #4 will define murine strain differences in response to VALI-mediated vascular leak and utilize experimental progeny backcross strategies with genetic mapping of the murine genome to identify QTLs linked to susceptibility to VALI. The Project Leader, an authority on molecular mechanisms of vascular barrier regulation, combines exceptional complementary skills and experts in animal models of lung injury and mouse genetics. Given the profound physiologic derangements which accompany the vascular leak seen in VALI, we speculate that this project which will explore novel therapies and targets for VALI-induced pulmonary edema, will more quickly allow us to bridge the movement of scientific discovery into direct benefit for patients with Acute Lung Injury.